Outward ionic binding modes and PMF.

<p>A) Overlay of the conformational space of the probe ions (yellow), coupling sodium ions (blue) and E53 carboxyl oxygen distributions (transparent spheres with different intensity) for different ionic binding modes at different interaction sites. A snapshot of a typical E53 sidechain conformation is shown as stick representation (red, carboxyl oxygens; yellow, sidechain carbons), I–V are non-flip binding modes and II′–IV′ are flipping binding modes for sites S_HFS, S_BAR and S_CEN. Arrows indicate the direction of ion conduction; B) 1-D PMF of outward conduction in SF region, the largest free energy barriers are labeled by terminal peaks and wells, corresponding positions of typical binding modes on the energy profile are labeled. Error estimations shown in the figure are S.E.M.</p

Annotations of the SF ion interacting site.

<p>SF backbone atoms are shown as blue sticks, E53 and S54 side chain atoms are also shown to determine the site S_HFS (only two opposite subunits are shown for clarity). In addition, sodium ions are depicted with yellow spheres. Sites S_EX (−5.00≤Z<0.00 Å), S_HFS (0.00≤Z<2.75 Å), S_BAR (2.75≤Z<4.75 Å), site S_CEN (4.75≤Z<7.75 Å) and site S_IN (7.75≤Z<10.25 Å) and the length of each site were annotated at the lateral sides of the figure.</p

Different Inward and Outward Conduction Mechanisms in Na_VMs Suggested by Molecular Dynamics Simulations

<div><p>Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. Here, we use molecular dynamics (MD) simulations with an applied membrane potential to investigate the ion flux of bacterial sodium channel Na_VMs. 5.9 µs simulations with 500 mM NaCl suggest different mechanisms for inward and outward flux. The predicted inward conductance rate of ∼27±3 pS, agrees with experiment. The estimated outward conductance rate is 15±3 pS, which is considerably lower. Comparing inward and outward flux, the mean ion dwell time in the selectivity filter (SF) is prolonged from 13.5±0.6 ns to 20.1±1.1 ns. Analysis of the Na⁺ distribution revealed distinct patterns for influx and efflux events. In 32.0±5.9% of the simulation time, the E53 side chains adopted a flipped conformation during outward conduction, whereas this conformational change was rarely observed (2.7±0.5%) during influx. Further, simulations with dihedral restraints revealed that influx is less affected by the E53 conformational flexibility. In contrast, during outward conduction, our simulations indicate that the flipped E53 conformation provides direct coordination for Na⁺. The free energy profile (potential of mean force calculations) indicates that this conformational change lowers the putative barriers between sites S_CEN and S_HFS during outward conduction. We hypothesize that during an action potential, the increased Na⁺ outward transition propensities at depolarizing potentials might increase the probability of E53 conformational changes in the SF. Subsequently, this might be a first step towards initiating slow inactivation.</p></div

Influences of different flipping states at Δq = 4<i>e</i> (n = 4).

<p>A) Average ion flux counts through the SF over time of inward conduction (color: blue). The simulations without restraints of E53 are depicted as solid line, the ones with the “one-flip” restraints are shown as dotted line and the ones with the “non-flip” restraints are shown as dashed line. B) Average ion flux count through the SF over time of outward conduction (color: red). Error estimations shown in the figure are S.E.M.</p

Ion distribution probability density map.

<p>A) Ion distribution of inward simulations labeled with respective interacting sites <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003746#pcbi.1003746-Payandeh1" target="_blank">[4]</a>, lower left: 2-D (axial and radial distribution) density; lower right: 1-D axial distribution along the pore axis; upper left: 1-D radial distribution from the center of the pore axis. B) Inward dwell time in respective interacting sites (158 ion conduction events from four simulations were taken for analysis). C) Ion distribution of outward simulations labeled with respective interacting sites <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003746#pcbi.1003746-Payandeh1" target="_blank">[4]</a>, lower left: 2-D (axial and radial distribution) density; lower right: 1-D axial distribution along the pore axis; upper left: 1-D radial distribution from the center of pore axis. D) Outward dwell time in respective interacting sites (79 ion conduction events from four simulations were taken for analysis).</p

Transmembrane voltages comparison.

<p>A) Electrostatic potential across the simulation box calculated from the simulations without dihedral restraints on E53. B) Electrostatic potential across the simulation box calculated from the snapshots (t>5 ns) extracted from the simulation in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003746#pcbi-1003746-g002" target="_blank">Figure 2</a>A without ion conduction events in neither inward nor outward directions. C) Electrostatic potential across the simulation box calculated from the “no salt” simulations. (Error estimations shown in the figure are S.D.).</p

Ion flux in double bilayer simulations without dihedral restraints on E53 (n = 4).

<p>A) Average ion flux count of inward conduction through the SF over time (color: blue). B) Average ion flux count of outward conduction through the SF over time (color: red). Error estimations shown in the figure are S.E.M.</p

PMF comparison for outward conduction.

<p>A) PMF of the simulations without restraints of E53 is depicted as solid line. (B) PMF of the simulations with “one-flip” restraints. C) PMF of the simulations with “non-flip” restraints. The largest free energy barriers in each figure are labeled by terminal peaks and wells. Error estimations shown in the figure are S.E.M.</p